LncRNA ZFAS1 regulates ATIC transcription and promotes the proliferation and migration of hepatocellular carcinoma through the PI3K/AKT signaling pathway

Purpose Long noncoding RNAs (lncRNAs) exert a significant influence on various cancer-related processes through their intricate interactions with RNAs. Among these, lncRNA ZFAS1 has been implicated in oncogenic roles in multiple cancer types. Nevertheless, the intricate biological significance and underlying mechanism of ZFAS1 in the initiation and progression of hepatocellular carcinoma (HCC) remain largely unexplored. Methods Analysis of The Cancer Genome Atlas Program (TCGA) database revealed a notable upregulation of lncRNA ZFAS1 in HCC tissues. To explore its function, we investigated colony formation and performed CCK-8 assays to gauge cellular proliferation and wound healing, Transwell assays to assess cellular migration, and an in vivo study employing a nude mouse model to scrutinize tumor growth and metastasis. Luciferase reporter assay was used to confirm the implicated interactions. Rescue experiments were conducted to unravel the plausible mechanism underlying the activation of the PI3K/AKT pathway by lncRNAs ZFAS1 and ATIC. Results ZFAS1 and ATIC were significantly upregulated in the HCC tissues and cells. ZFAS1 knockdown inhibited cell proliferation and migration. We observed a direct interaction between the lncRNA ZFAS1 and ATIC. ATIC knockdown also suppressed cell proliferation and migration. SC79, an activator of AKT, partially restores the effects of lncRNA ZFAS1/ATIC knockdown on cell proliferation and migration. Knockdown of lncRNA ZFAS1/ATIC inhibited tumor growth and lung metastasis in vivo. Conclusion Overall, lncRNA ZFAS1 regulates ATIC transcription and contributes to the growth and migration of HCC cells through the PI3K/AKT signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-024-05877-1.


Introduction
Hepatocellular carcinoma (HCC) is a highly prevalent neoplasm that significantly contributes to cancer-related mortality worldwide (Siegel et al. 2017).Despite remarkable advancements in diagnostic techniques and surgical procedures, and the advent of novel molecular targeted therapies, HCC remains the primary cause of cancer-related fatalities globally, accounting for a staggering 600 thousand annual deaths (Ma et al. 2017;Omata et al. 2017).Unfortunately, the current surveillance and treatment approaches fail to meet satisfactory standards.A substantial risk factor affecting the prognosis of HCC is the high incidence of tumor recurrence and distant metastasis after hepatectomy (Pi et al. 2016).Therefore, it is imperative to elucidate the mechanisms underlying the initiation and metastasis of HCC to enhance its early detection and prognostic capabilities.
Long noncoding RNAs (lncRNAs) are RNA molecules that exceed 200 bp and do not possess the capacity to encode proteins (Fang et al. 2016).LncRNAs play a crucial role in diverse biological processes including autophagy (Liang et al. 2020), proliferation (Hu et al. 2017;Luo et al. 2019) and metastasis (Abbastabar et al. 2018) of cancer cells.Furthermore, lncRNAs orchestrate tumorigenesis and metastasis in diverse malignancies, including HCC, by perturbing gene expression at the transcriptional or epigenetic level (Abbastabar et al. 2018;Yao et al. 2022).Multiple lncRNAs have been reported to regulate lung cancer growth and metastasis (Huang et al. 2021b(Huang et al. , 2024)).The lncRNA ANCR exhibited heightened expression in both HCC tissues and cells, displaying a positive association with tumor differentiation, TNM stage, tumor size, and the presence of portal vein tumor thrombus (Wen et al. 2020).Similarly, the lncRNA ALKBH3-AS1 displays heightened expression in HCC, with its elevated levels notably linked to reduced survival among HCC patients (Lu et al. 2022).Another newly discovered lncRNA, ZFAS1, has been found to exhibits increased expression in diverse human malignancies, including colorectal cancer (Wang et al. 2022), HCC (Zhu et al. 2023a), pancreatic cancer (Zhuo et al. 2023) and laryngeal cancer (Lu et al. 2023).The lncRNA ZFAS1 contributes to the progression of this disease through the miR-624/ MDK/JNK/ERK/P38 pathway (Duan et al. 2020a).However, the exact biological function of ZFAS1 and its underlying molecular mechanisms, particularly in HCC, remain largely unknown.
In this study, we analyzed the expression of disease-associated lncRNAs in HCC tissues.Remarkably, we discovered that lncRNA ZFAS1 exhibited an upregulated expression pattern, specifically within HCC tissues.Subsequently, we investigated the effects of ZFAS1 expression on crucial cellular processes.Moreover, we explored the potential interactions between the lncRNA ZFAS1 and the gene to elucidate their involvement in the functional mechanisms of ZFAS1.

Bioinformatics analysis
RNA-Seq data were obtained from the Cancer Genome Atlas (TCGA) database, encompassing a comprehensive set of 50 normal samples and 373 HCC samples.Differential expression analysis was conducted using the limma package in R4.1.2,employing a stringent threshold of FDR < 0.05 and |log 2 FC|>1 to identify significantly differentially expressed genes (DEGs) and lncRNAs (DE-lncRNAs).To illustrate the differences in gene expression between tumor and control samples, we utilized the Pheatmap version 1.0.8package in R4.1.2,to perform bidirectional hierarchical clustering according to the expression profiles of DE-lncRNAs and DEGs.The Pearson Correlation Coefficient (PCC) between DE-lncRNAs and DEGs was computed using the COR function in R4.1.2language.Subsequently, by selecting connection pairs with a PCC > 0.6 and P < 0.05, we constructed and visualized a co-expression network of DE-lncRNAs and DEGs using Cytoscape version 3.6.1.
Furthermore, patients with HCC were categorized into high-and low-expression groups of lncRNA ZFAS1 based on their expression levels.Fisher's exact test was used to assess the clinical relationship between ZFAS1 expression and age, sex, neoplasm histologic grade, pathologic M, pathologic N, pathologic T, pathologic stage, recurrence, and vital status.

Cell culture
The HepG2, Hep3B, and WRL68 cell lines derived from human hepatocellular carcinoma, were acquired from the Cell Bank of the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China).The cells were cultured in Modified Eagle Medium (MEM) (Servicebio, China) supplemented with 1% penicillin/streptomycin (Gibco, USA) and 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in a humidified incubator with 5% CO 2 .

Patient samples collection
Five paired HCC tissue samples and their corresponding adjacent normal tissues were obtained from patients who underwent surgical resection between February 2022 and May 2022 at our hospital.Resected tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C 1 3 before RNA extraction.This study was approved by the Ethics Committee of the Affiliated Taizhou People's Hospital of Nanjing Medical University, and written informed consent was obtained from all patients.

Cell transfection
Small interfering RNA (siRNA) molecules designed to target lncRNA ZFAS1 (si-lncRNA ZFAS1) and their corresponding control (si-NC), as well as siRNA targeting ATIC (si-ATIC) and its matched control (si-NC), were synthesized by Shanghai Generay Biotech Co., Ltd.(Shanghai, China).Prior to transfection, cells were cultured in a complete medium for a minimum of 24 h.For cell transfection, Lipofectamine 2000 (Thermo Fisher Scientific) was used in accordance with the manufacturer's instructions to ensure efficient delivery of siRNA molecules into the cells.

Real time-polymerase chain reaction (RT-PCR)
TRIzol reagent (Thermo Fisher Scientific) was used to extract total RNA from the cells.Subsequently, the extracted total RNA was reverse-transcribed using a PrimeScript RT Reagent Kit (Takara, Japan) following the manufacturer's instructions.RT-qPCR was performed using a SYBR Green PCR Kit (Thermo Fisher Scientific).The RT-qPCR program commenced with an initial step at 95 °C for 2 min, succeeded by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s.The expression levels of the target genes were normalized to the reference gene GAPDH and calculated utilizing the 2 −ΔΔCT method.The primer sequences used in this study are listed in Table 1.

CCK-8 assay
Cell viability was assessed using the CCK-8 assay (Beyotime, China).Transfected cells were resuspended at a concentration of 1 × 10 4 cells/mL and plated in 96-well plates.Following 24, 48, and 72 h of incubation, the cells were treated with CCK-8 (10 µL) for 2 h.Absorbance at 450 nm was measured using a microplate reader.

Colony formation assay
The cells were seeded into a 6-well plate and subjected to a two-week cultivation until discernible cell colonies became visible to the naked eye.Subsequently, the cells were rinsed and fixed in 4% paraformaldehyde solution for 10 min.Following fixation, staining was performed using 1% crystal violet solution (Beyotime, China) for 10 min.After drying, the colonies were counted under a microscope to determine the colony formation rate.

Transwell migration assay
Cell migration was investigated using transwell chambers (Guangzhou Jet Bio-Filtration Co., Ltd., China).Briefly, cells subjected to different treatments were seeded in the upper compartment of the transwell chambers, while the lower compartment was supplemented with complete medium.After 48 h of incubation at 37 °C, the cells were immobilized in a 4% paraformaldehyde solution for 20 min.Subsequently, the cells were stained with 1% crystal violet for 20 min.Cell migration was visualized under a microscope, and the number of migrated cells was quantified.

Wound healing assay
Migratory potential was evaluated using a wound healing assay.Initially, a marker pen was used to delineate a straight line on the surface of the 6-well plates.The transfected cells were then seeded in these plates and cultured until they reached 80-90% confluence.Subsequently, two parallel lines were inscribed perpendicularly to the initial marking line using a pipette tip.The scratched cells were then washed with PBS, and each well was filled with serum-free medium.Images of the cellular landscape were captured at the initiation of the assay and after a 48-hour duration, utilizing an inverted microscope.

Dual-luciferase reporter assay
The cells were carefully distributed in a 96-well plate.At 48 h post-transfection, luciferase activity was quantified using a dual-luciferase reporter gene assay kit (Beyotime) according to the guidelines provided by the manufacturer's instructions.To ensure consistency and accuracy, the data obtained were normalized by calculating the ratio of firefly to Renilla luciferase activity.

Western blot analysis
RIPA buffer (Beyotime, China) was used to lyse the total proteins extracted from HCC cells.The BCA Protein Assay alcohol solution.The sections were then stained with an aqueous solution of eosin for 90 s, dehydrated using a series of alcohol gradients, and rendered transparent with xylene.

Immunofluorescence
The tumor and lung tissues were fixed in 4% paraformaldehyde, followed by dehydration with sucrose.Subsequently, the tissues were embedded in optimal cutting temperature (OCT) compound and cryopreserved at -80 °C for 1 h.Then, they were sectioned into 10 μm thick sections.The frozen sections were baked in a 37 °C oven for 20 min to remove excess moisture.After drying, the sections were mounted with an anti-fluorescence quenching mounting medium (containing DAPI) and covered with a coverslip.Observations and photographs were obtained using a confocal laser microscope.

Immunohistochemistry (IHC)
For IHC staining, paraffin-embedded tissue sections were subjected to antigen retrieval using citrate buffer, and the activity of endogenous peroxidase was inhibited using a 3% hydrogen peroxide solution.Subsequently, the sections were rinsed with phosphate-buffered saline and incubated with a primary antibody against ATIC (1:100 dilution, Proteintech, USA) for 1 h at 37 °C.Subsequently, the sections were subjected to a 30-min incubation with a secondary antibody labeled with horseradish peroxidase (HRP), followed by visualization using amine nickel sulfate-enhanced 3,3'-diaminobenzidine.The nuclei were stained with hematoxylin, and images were taken under a light microscope at 200x magnification.

Statistical analysis
The data were subjected to analysis using GraphPad Prism 8.0 software and were expressed as the mean ± standard deviation (SD).T-tests were used to assess the differences between two groups, whereas one-way analysis of variance (ANOVA) was used to compare the differences among three or more groups.A significance level of P < 0.05 was considered statistically significant.

Bioinformatics prediction of lncRNA ZFAS1 and targeting mRNA and validation
Initially, we identified 1387 differentially expressed RNAs between HCC and control tissues using TCGA dataset.

Animal experiments
The animal experimental protocols were approved by the Committee for Ethical Review of Research of the Affiliated Wujin People's Hospital of Jiangsu University (Protocol Number: SYXK 2018-0053).A total of nine male BALB/c-nu/nu nude mice, aged 4-6 weeks, were acquired from Shanghai SLAC Laboratory Animal Co., Ltd.(Shanghai, China).Following transfection with si-NC, si-lncRNA ZFAS1, or si-ATIC, cells were thoroughly washed with PBS.Subsequently, transfected cells were suspended at a density of 5 × 10 7 cells/ml.The mice were then randomly assigned to three groups and injected with 100 µl of cell suspension into the forelimb through the axilla.The weight of the mice and length of the tumors were assessed weekly.After 4 weeks, the nude mice were humanely euthanized, and the bioluminescence emitted from the mouse body was collected using an IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer).The tumors were carefully peeled off and weighed.Tumor and lung tissues were collected and meticulously preserved in 4% paraformaldehyde solution for fixation.

Hematoxylin and eosin (H&E) staining
Paraformaldehyde-fixed tissues were dehydrated through a succession of alcohol gradients and subsequently embedded in paraffin.Subsequently, paraffin-embedded sections were deparaffinized using xylene and rehydrated using an ethanol gradient.10-min staining procedure with hematoxylin ensued, followed by immersion in 1% hydrochloric acid 1 3 linked to the development and progression of the disease (Abozeid et al. 2022;Duan et al. 2020b;Zhou et al. 2019).Therefore, we selected lncRNA ZFAS1 as the target of our study.In addition, we found that the lncRNA ZFAS1 interacted with ATIC in the ceRNA network and that ATIC was highly expressed in HCC tissues (Fig. 2C).Furthermore, ATIC overexpression was linked to poor disease-free survival and overall survival in patients with HCC (Fig. 2D).
To validate these findings, we used RT-PCR to assess the expression levels of ZFAS1 and ATIC in HCC and control tissues.The results demonstrated a notable upregulation of both lncRNA ZFAS1 and ATIC in HCC tissues compared to control tissues (Fig. 2E, P < 0.05).Consequently, we hypothesized that lncRNA ZFAS1 plays a crucial role Among these, there were 24 DE-lncRNAs with 9 downregulated and 15 upregulated genes and 1363 DEGs with 383 downregulated genes and 980 upregulated genes (Fig. 1A).
Hierarchical clustering analysis revealed systematic variations in lncRNA expression patterns between HCC and control tissues (Fig. 1B).Subsequently, using a predefined threshold, we identified 450 regulatory pairs co-expressed between the DE lncRNAs and DEGs.Based on these regulatory pairs, we established a co-expression network comprising 14 DE lncRNAs and 257 DEGs (Fig. 1C).Among them, lncRNA ZFAS1 exhibited significantly higher expression levels in HCC tissues than in control tissues (Fig. 2A), and lncRNA ZFAS1 overexpression was linked to poor disease-free survival and poor overall survival in HCC patients (Fig. 2B).LncRNA ZFAS1 is upregulated in HCC and is had a higher proportion of patients with neoplasm histologic grade 3, neoplasm histologic grade 4, and mortality, whereas fewer patients had neoplasm histologic grades 3 and 4 (Table 2, P < 0.05).However, no significant differences were observed between the two groups in terms of age, sex, pathologic M, pathologic N, pathologic T, pathologic stage, or recurrence (Table 2; P > 0.05).
in promoting HCC progression through its interaction with ATIC.

Relationship between lncRNA ZFAS1 expression and clinical factors
Subsequently, the relationship between lncRNA ZFAS1 expression and clinical factors was analyzed.The results indicated that compared to the low lncRNA ZFAS1 expression group, the high lncRNA ZFAS1 expression group As depicted in Fig. 4E, the expression of ATIC was significantly decreased in the siRNA-ATIC-1, siRNA-ATIC-2, and siRNA-ATIC-3 groups compared to that in the si-NC group.As siRNA-ATIC-1 had the highest knockout efficiency in HepG2 cells, siRNA-ATIC-1 was used for follow-up experiments.Western blotting revealed significantly lower ATIC expression in the si-ATIC group than in the blank and si-NC groups (Fig. 4F, P < 0.05).CCK-8 and colony formation assays revealed that si-ATIC significantly inhibited HCC cell proliferation compared to that in the blank and si-NC groups (Fig. 4F, G, P < 0.05).Furthermore, transwell migration and wound healing assays revealed that ATIC knockdown significantly inhibited the migration of HCC cells compared to the control and si-NC groups (Fig. 4H, I, P < 0.05).These results confirmed that knockdown of either ATIC or lncRNA ZFAS1 exerts a similar inhibitory effect on HCC proliferation and metastasis.

LncRNA ZFAS1-ATIC axis regulated proliferation and migration via PI3K/AKT pathway
The PI3K/AKT pathway is widely recognized as a pivotal oncogenic pathway in diverse types of cancers, including HCC (Paskeh et al. 2023;Vara et al. 2004).Moreover, the PI3K/AKT pathway plays a crucial role in governing fundamental biological functions such as cell proliferation and metabolism.Therefore, we investigated the effect of the ZFAS1-ATIC axis on the PI3K/AKT pathway in HCC.
To test this hypothesis, western blotting was performed to assess the protein levels of p-PI3K, PI3K, AKT, and p-AKT.Remarkably, our findings demonstrated that treatment with si-lncRNA ZFAS1 and si-ATIC resulted in decreased expression of p-PI3K and p-AKT compared to that in the blank and si-NC groups (Fig. 5).
To further investigate whether the lncRNA ZFAS1-ATIC axis regulates HCC migration and proliferation through the PI3K/AKT pathway, a series of rescue experiments were conducted using SC79, an AKT activator.Initially, we observed that cell proliferation was not inhibited with increasing concentrations of SC79 from 0 to 2 µg/mL (Fig. 6A).However, cell viability was induced in a concentration-dependent manner upon treatment with SC79 (4, 8, 16, 32, 64 µg/mL).Consequently, 2 µg/mL SC79 was utilized for further experiments.As illustrated in Fig. 6B, C,  D, and E, CCK-8, colony formation, transwell migration, and wound healing assays revealed that the cells exhibited significantly augmented cellular proliferation and migration capabilities compared to the si-lncRNA ZFAS1 group.Similarly, the si-ATIC + SC79 group exhibited significantly enhanced cell proliferation and migration compared to the si-ATIC group (Fig. 6B, C, D, and E, P < 0.05).These findings

LncRNA ZFAS1 and ATIC expression increased in HCC cell lines
ZFAS1 and ATIC expression levels were assessed in various HCC cell lines (Hep3B, HepG2, and WRL68).Remarkably, lncRNAs ZFAS1 and ATIC exhibited pronounced upregulation in HepG2 and Hep3B cells compared to WRL68 cells, with HepG2 cells demonstrating particularly elevated expression levels (Fig. 2F, G, P < 0.05).Consequently, HepG2 cells were selected for subsequent experiments.

Knockdown of lncRNA ZFAS1 inhibited proliferation and migration of HCC cells
To study the impact of lncRNA ZFAS1 on HCC cells, we knocked down lncRNA ZFAS.The knockdown efficiency was assessed using RT-PCR, and it was observed that the three siRNAs effectively downregulated lncRNA ZFAS1, with si-lncRNA ZFAS1-2 exhibiting the highest knockdown efficiency (Fig. 3A).Subsequently, a series of assays were performed to evaluate the cell proliferation and migration capabilities of HCC cells.CCK-8 assays indicated that si-lncRNA ZFAS1 significantly inhibited HepG2 cell viability (Fig. 3B).Similarly, colony formation assays revealed that si-lncRNA ZFAS1 markedly suppressed the clonogenic potential of HepG2 cells (Fig. 3C).Moreover, transwell and wound healing assays indicated that si-lncRNA ZFAS1 notably inhibited the migratory capacity of the cells (Fig. 3D, E).These results collectively implied that knockdown of lncRNA ZFAS1 inhibited the growth and migration of HCC cells.

ATIC participated in the process of lncRNA ZFAS1 mediated proliferation and migration
Analysis using the Miranda v3.3a software revealed a binding interaction between the upstream region of the ATIC gene transcription start site and lncRNA ZFAS1 (Table S1).To further explore this interaction, we cloned the region upstream of the ATIC transcription start site into a pGL3 luciferase vector and transfected it into si-NC and si-lncRNA cells.As depicted in Fig. 4A, the knockdown of ZFAS1 resulted in a decline in luciferase activity.TCGA database revealed a positive association between the expression of lncRNA ZFAS1 and ATIC in HCC tissues (Fig. 4B).Furthermore, RT-PCR and western blotting results demonstrated a notable reduction in ATIC expression in the si-lncRNA ZFAS1 group compared to the blank and si-NC groups (Fig. 4C, D).These findings indicate that lncRNA ZFAS1 can activate ATIC gene transcription.
Subsequently, ATIC knockdown experiments were performed to verify the role of ATIC in HCC development.in the number of green fluorescent cells in tumor tissues between the si-lncRNA ZFAS1 group and si-ATIC group compared to that in the control group (Fig. 8A, P > 0.05).However, the number of green fluorescent cells was significantly diminished in the lung tissues of both the si-lncRNA ZFAS1 and si-ATIC groups compared to that in the control group (Fig. 8B, P < 0.05).

Discussion
HCC is a widely recognized malignancy worldwide and is the third most prevalent cause of cancer-associated mortality.HCC is the main histological subtype of primary liver cancers and accounts for 70% of the worldwide burden of liver cancer (Jemal et al. 2011;Perz et al. 2006).The insidious onset and lack of early clinical symptoms make the early detection and diagnosis of HCC challenging (Zong et al. 2020).Typically, HCC is diagnosed at advanced stages, contributing to its persistently high mortality rates owing to its propensity for invasion and metastasis indicate that the lncRNA ZFAS1-ATIC axis promotes HCC progression by modulating the PI3K/AKT pathway.

Knockdown of lncRNA ZFAS1 and ATIC suppresses tumor growth and metastasis in nude mice
To validate the inhibitory effects of ZFAS1 and ATIC in vivo, HepG2 cells with si-lncRNA ZFAS1 or si-ATIC and injected into nude mice.Notably, Fig. 7A, B, C demonstrate a substantial reduction in tumor volume in the si-lncRNA ZFAS1 and si-ATIC groups, in contrast to the control group, after 4-week.The In Vivo Imaging System detected a significant decrease in the intensity of green fluorescence emitted by the si-lncRNA ZFAS1 and si-ATIC groups compared to that in the control group (Fig. 7D).Morphological alterations were observed in the si-lncRNA ZFAS1, si-ATIC, and control groups by histological examination using HE staining (Fig. 7E).IHC staining revealed decreased ATIC expression in the si-lncRNA ZFAS1 and si-ATIC groups relative to the control group (Fig. 7F, P < 0.05).Moreover, the immunofluorescence results revealed no significant differences and modulation of transcriptional activation or interference.ZFAS1, an intriguing lncRNA transcribed in the opposite orientation to the NFX1 zinc finger gene, resides on the 20q13.13locus of the human chromosome.Initially, ZFAS1, a novel tumor-associated lncRNA, was found to enhance epithelial-mesenchymal transition, migration, and cell proliferation (Askarian-Amiri et al. 2011).Recently, the involvement of LncRNA-ZFAS1 in the pathogenesis of various cancers has been extensively reported (Chen et al. 2018;Pan et al. 2020;Xu et al. 2018).For instance, Zhang et al. showed that lncRNA ZFAS1 curtails the migration, invasion, and growth of breast cancer cells by modulating the PTEN/PI3K/AKT pathway via its interaction with miR-589 (Zhang et al. 2020).Liu et al. demonstrated that ZFAS1 promotes metastasis in pancreatic adenocarcinoma through the RHOA/ROCK2 pathway by acting as a miR-3924 sponge (Liu et al. 2020).In HCC, ZFAS1 is an lncRNA capable of influencing disease progression through diverse regulatory pathways.Zhang et al. substantiated the direct interaction between lncRNA ZFAS1 and miR-150-5p, which exerts a profound influence on the migration, proliferation, and invasion of HCC cells (Zhu et al. 2023b).Similarly, the (Chidambaranathan-Reghupaty et al. 2021).Consequently, there is an urgent need to identify valuable molecular markers for the early detection and identification of therapeutic targets for the treatment of HCC.In this study, we revealed that lncRNA ZFAS1 and ATIC were highly expressed in HCC tissues, and that lncRNA ZFAS1 could directly bind to ATIC to form the lncRNA ZFAS1-ATIC axis.Furthermore, the knockdown of lncRNA ZFAS1 and ATIC markedly suppressed the growth and migratory capabilities of HCC cells.Further mechanistic studies showed that the ZFAS1-ATIC axis regulates the biological role of HCC cells by activating the PI3K/AKT signaling pathway.Furthermore, we verified that the knockdown of ZFAS1 and ATIC inhibited HCC growth in vivo in a nude mouse transplantation model.In summary, our study revealed the important role and molecular mechanism of the lncRNA ZFAS1-ATIC axis in HCC, providing new targets and strategies for the treatment of HCC (Fig. 9).
LncRNAs, a distinct category of non-coding RNAs, exert a crucial influence on gene expression modulation through diverse mechanisms, including chromatin structure remodeling (Huang et al. 2021b), DNA methylation regulation, ATIC is a bifunctional enzyme with a molecular weight of 64 kDa that regulates the activities of two enzymes involved in de novo purine biosynthesis, IMP cyclohydrolase and AICAR (Greasley et al. 2001).There is a correlation between ATIC and rheumatoid arthritis, tumor cell proliferation, and the effectiveness of radiotherapy (Liu et   lung metastasis.These findings highlight the involvement of ATIC in the functional modulation of HCC cells through the mediation of lncRNA ZFAS1. The PI3K/AKT pathway is a critical intercellular signaling cascade that is pivotal in driving cancer progression, metastasis, and metabolic alterations (Huang et al. 2021a).The PI3K/AKT pathway has been known to be activated in various tumors, including HCC (Wu et al. 2020).Recent studies have shed light on the role of lncRNAs in regulating the PI3K/AKT pathway.For example, Wang et al. discovered that lncRNA ZFAS1 stimulates proliferation and migration while suppressing apoptosis in nasopharyngeal cancer via the activation of the PI3K/AKT pathway (Wang et al. 2019b).Similarly, Wang et al. proposed that the upregulation of the lncRNA FER1L4 exerts a suppressive effect on the migration and proliferation of HCC cells by modulating the PI3K/AKT signaling pathway (Wang et al. 2019a).Furthermore, Zheng et al. found that LINC01133 knockdown significantly impedes HCC tumor development by targeting the PI3K/AKT pathway (Zheng et al. 2019).SC79 functions as an activator of AKT, eliciting the phosphorylation of AKT to initiate activation of the PI3K/AKT signaling cascade (Luan et al. 2018).Here, we observed that SC79 partially restored the suppressive consequences of ZFAS1/ ATIC downregulation on HCC cell proliferation and migration.Consequently, we deduced that the ZFAS1-ATIC axis promotes HCC cell proliferation and migration by modulating the PI3K/AKT signaling pathway.
However, this study had some limitations.First, we only examined the ceRNA network that regulates lncRNA ZFAS1 in ATIC, while the other regulatory mechanisms mentioned above that may be involved require further investigation.Second, the specific miRNAs involved in the ZFAS1-ATIC axis have not been identified, and the potential upstream regulatory factors controlling this axis have not been explored.Therefore, further identification of specific miRNAs involved in the ZFAS1-ATIC axis and exploration of potential upstream regulatory factors controlling this axis is required.
In summary, we uncovered the pivotal significance of the lncRNAs ZFAS1 and ATIC in the progression of HCC.Our findings demonstrate that lncRNA ZFAS1 regulates ATIC transcription and promotes the proliferation and migration of HCC cells through the PI3K/AKT pathway.Moreover, the lncRNA ZFAS1-ATIC axis holds promise as both a diagnostic biomarker and therapeutic target for HCC.

Fig. 6
Fig. 6 SC79 (a AKT activator) partially restores long non-coding RNA (lncRNA) ZFAS1 and ATIC knockdown HepG2 cells.(A) CCK-8 assay results revealing the viability of HCC cells treated with different concentration of SC79.(B) CCK-8 assay results revealing the viability of HCC cells treated with different treatments.(C) Colony formation assay results revealing the proliferation of HCC cells treated with different treatments and colonies were counted and captured.(D) Transwell assay results revealing the migration of HCC cells treated with different treatments.(E) Wound healing assay results revealing the migration of HCC cells treated with different treatments.*P < 0.05, compared with si-lncRNA ZFAS1.# P < 0.05, compared with si-ATIC

Fig. 7
Fig. 7 Knocking down lncRNA ZFAS1 and ATIC could inhibit the growth of HCC in vivo.(A) HepG2 cells transfected with si-lncRNA ZFAS1 or si-ATIC were subcutaneously injected into the armpit of nude mice (n = 3 each group).Tumors before and after carrying from the nude mice.(B) Tumor volumes calculated after injection every week.(C) Tumor inhibition rate of each group.(D) Tumor sections with hematoxylin and eosin (H&E) staining.(E) Representative image and analysis of luminescence intensity in the mice models.(F) Tumor sections with immunohistochemistry (IHC) staining.*P < 0.05, compared with control group

Fig. 9 Fig. 8
Fig. 9 Regulatory mechanism of LncRNA ZFAS1 in modulating ATIC transcription and promoting the growth and migration of HCC through the PI3K/AKT signaling pathway is elucidated.In in vitro experiments showing the inhibition of HepG2 cell proliferation and migration upon

Table 1
The primer sequences used in this study

Table 2
Relationship between ZFAS1 expression and clinical factors